Phospholipases A.sub.2 constitute a diverse family of enzymes with two subclasses (Type I and Type II) (FIG. 1), based on the positions of the disulphide bonds in the molecules while bee venom PLA.sub.2 constitutes a third substantially distinct class of PLA.sub.2. X-ray crystallography has revealed that the segments comprising the functional substructure of the enzyme is similar in classes. This similarity is particularly striking when the structurally-related Type I/II enzymes are compared with bee venom enzyme (2). PLA.sub.2 hydrolyses the sn-2 acyl ester bond of phosphoglycerides initiating the release of fatty acid precursors of inflammatory eicosanoids. Human synovial PLA.sub.2 (a Type II molecule) has recently been isolated and identified (3). The same PLA 2 has been implicated in the pathogensis of several inflammatory diseases in humans such as rheumatoid arthritis and Gram negative septic shock (7;8).
Murine, inhibitory monoclonal antibodies raised against synovial PLA.sub.2 have demonstrated pre-clinical efficacy. Accordingly, there is interest in the development of compositions which inhibit the enzymatic activity of PLA.sub.2.
Tryptic digestion of human synovial PLA.sub.2 and subsequent separation and analysis of the fragments by EPLC gave a very interesting and unexpected result for one of the peaks in that it contained two peptides; one a heptapeptide (the N-terminal peptide) and the other a pentapeptide, FLSYK (SEQ ID NO:8) (corresponding to residues 70-74 in other PLA.sub.2 molecules, based on three-dimensional structural "homology" of mammalian PLA.sub.2 amino acid sequences (1,4)). The pentapeptide was found in an earlier eluting, fully resolved peak (as expected). Since the HPLC system failed to fully resolve these two peptides in the latter peak, these data suggest that the two peptides had a strong affinity for one another and raised questions as to the structural implications of this. X-ray diffraction studies (5,6) have shown that amino acid residues in the two peptides are close to the active site of the enzyme and are important in forming or stablising the channel in which the 1,2-diacyl-3-sn-phosphoglyceride substrate is precisely positioned for hydrolysis of the 2-ester bond. The first turn of the N-terminal helix (residues 1 to 12) is stablised by a hydrogen bond network provided by the N-terminus and residue 4, elements of residues 69 to 71 and a water mediated link to the catalytic residues; residues 2 and 5 form the "floor" of the channel, residue 9 forms the right wall and the left wall is formed by residue 69 (either tyrosine or lysine usually) which is predicted to move after the substrate has docked and to form a hydrogen bond with the sn-3 phosphate of the substrate. The chemical evidence of the strong interactions between the heptapeptide and the pentapeptide prompted the supposition that the PLA.sub.2 activity may be inhibited in the presence of either one of these peptides.
Using synthetic peptide chemistry the present inventors have prepared the pentapeptide FLSYK and demonstrated that addition of it to the assay medium decreased the enzyme activity of human synovial PLA.sub.2 (FIG. 2a). Furthermore, it has been demonstrated that the pentapeptide that occupies the 70-74 region of snake PLA.sub.2 (WDIYR) also inhibited the activity of snake PLA.sub.2 (see FIG. 3b). It is believed that this inhibition is mediated by the peptide binding to the amino terminal end of the enzyme and blocking the reaction either by blocking the substrate access to the hydrophobic channel or by distorting the structure sufficiently to prevent correct orientation of the substrate.